Molecular Mechanisms of Anesthetic Action
All general anesthetics in common clinical use modulate either three-transmembrane (TM3) ion channels (e.g., NMDA receptors), four-transmembrane (TM4) ion channels (e.g., GABAA receptors), or members of both ion channel superfamilies. Sonner, et al., Anesth Analg (2003) 97:718-40. For example, many structurally unrelated inhaled anesthetics potentiate GABAA currents and inhibit NMDA currents. But why should a diverse group of compounds all modulate unrelated ion channels? A highly specific “induced fit” model between protein and ligand, as proposed for enzyme-substrate binding, (Koshland, Proc Natl Acad Sci USA 1958; 44: 98-104) is problematic since it implies the conservation of specific binding sites across non-homologous proteins to compounds (i.e., anesthetics) not found in nature. Sonner, Anesth Analg (2008) 107: 849-54. Moreover, promiscuous anesthetic actions on disparate receptors typically occurs at drug concentrations 50-200 times the median effective concentration (EC50) at which modulation of a single receptor class typically occurs, such as with etomidate agonism of GABAA receptors (Tomlin et al., Anesthesiology (1998) 88: 708-17; Hill-Venning, et al., Br J Pharmacol (1997) 120: 749-56; Belelli, et al., Br J Pharmacol (1996) 118: 563-76; Quast, et al., J Neurochem (1983) 41:418-25; and Franks, Br J Pharmacol 2006; 147 Suppl 1: S72-81) or dizocilpine (MK-801) antagonism of NMDA receptors. Wong, et al., Proc Natl Acad Sci USA (1986) 83: 7104-8; Ransom, et al., Brain Res (1988) 444: 25-32; and Sircar, et al., Brain Res (1987) 435: 235-40. It is unknown what molecular properties confer specificity for a single receptor (or members of a single receptor superfamily) and what properties allow other anesthetics to modulate multiple unrelated receptors. However, since ion channel modulation is important to conferring desirable anesthetic efficacy—as well as undesirable drug side effects—it is desirable to know what factors influence anesthetic receptor specificity in order to develop new and safer agents.
Anesthetics and Specific Ion Channel Targets
General anesthetics mediate central nervous system depression through actions on cell membrane receptors and channels which have a net hyperpolarizing effect on neurons. Sonner, et al., Anesth Analg (2003) 97:718-40; Grasshoff, et al., Eur J Anaesthesiol (2005) 22: 467-70; Franks, Br J Pharmacol (2006) 147 Suppl 1: S72-81; 33; Hemmings, et al., Trends Pharmacol Sci (2005) 26: 503-10; and Forman, et al., Int Anesthesiol Clin (2008) 46: 43-53. Although anesthetics partition into cell membranes as a function of lipid solubility, it is through competitive protein binding that these agents most likely produce anesthetic effects. In fact, general anesthetics have been shown to competitively inhibit functions of membrane-free enzymes (Franks, et al., Nature (1984) 310: 599-601), indicating that the lipid phase is not essential for anesthetic modulation of protein function. Specific high-affinity binding sites have been identified for some of these anesthetics. For example, apropofol (Jewett, et al., Anesthesiology (1992) 77: 1148-54; Bieda, et al., J Neurophysiol (2004) 92: 1658-67; Peduto, et al., Anesthesiology 1991; 75: 1000-9; Sonner, et al, Anesth Analg (2003) 96: 706-12; and Dong et al., Anesth Analg (2002) 95: 907-14), etomidate (Flood, et al., Anesthesiology (2000) 92: 1418-25; Zhong, et al., Anesthesiology 2008; 108: 103-12; O'Meara, et al., Neuroreport (2004) 15: 1653-6), and thiopental (Jewett, et al., Anesthesiology (1992) 77: 1148-54; Bieda, et al, J Neurophysiol (2004) 92: 1658-67; Yang, et al., Anesth Analg (2006) 102: 1114-20) all potently potentiate GABAA receptor currents, and their anesthetic effects are potently antagonized or prevented by GABAA receptor antagonists, such as pictotoxin or bicuculline. Ketamine produces anesthesia largely (but not entirely) through its antagonism of NMDA receptors. Harrison et al., Br J Pharmacol (1985) 84: 381-91; Yamamura, et al., Anesthesiology (1990) 72: 704-10; and Kelland, et al., Physiol Behav (1993) 54: 547-54. Dexmedetomidine is a specific a2 adrenoreceptor agonist that is antagonized by specific a2 adrenoreceptor antagonists, such as atipamezole. Doze, et al., Anesthesiology (1989) 71: 75-9; Karhuvaara, et al., Br J Clin Pharmacol (1991) 31: 160-5; and Correa-Sales, et al., Anesthesiology (1992) 76: 948-52. It is probably not by coincidence that anesthetics for which a single receptor contributes to most or all of the anesthetic effect also have low aqueous ED50 values (see, Table 1).
TABLE 1Aqueous phase EC50 for several anesthetics.AqueousAnestheticEC50 (μM)SpeciesReferencePropofol2RatTonner et al., Anesthesiology(1992) 77: 926-31Ketamine2HumanFlood, et al., Anesthesiology(2000) 92: 1418-25Etomidate3TadpoleTomlin, et al., Anesthesiology(1998) 88: 708-17Dexmedetomidine7TadpoleTonner, et al., Anesth Analg(1997) 84: 618-22Thiopental25HumanFlood, et al., Anesthesiology(2000) 92: 1418-25Methoxyflurane210TadpoleFranks, et al., Br J Anaesth(1993) 71: 65-76Halothane230TadpoleFranks, et al., Br J Anaesth(1993) 71: 65-76Isoflurane290TadpoleFranks, et al., Br J Anaesth(1993) 71: 65-76Chloroform1300TadpoleFranks, et al., Br J Anaesth(1993) 71: 65-76Diethyl ether25000TadpoleFranks, et al., Br J Anaesth(1993) 71: 65-76
Ion channel mutations, either in vitro or in vivo, dramatically alter anesthetic sensitivity, not only for the very potent and specific agents, but also for the inhaled anesthetics. Several mutations in the GABAA (Hara, et al., Anesthesiology 2002; 97: 1512-20; Jenkins, et al., J Neurosci 2001; 21: RC136; Krasowski, et al., Mol Pharmacol 1998; 53: 530-8; Scheller, et al., Anesthesiology 2001; 95: 123-31; Nishikawa, et al., Neuropharmacology 2002; 42: 337-45; Jenkins, et al., Neuropharmacology 2002; 43: 669-78; Jurd, et al., FASEB J 2003; 17: 250-2; Kash, et al., Brain Res 2003; 960: 36-41; Borghese, et al., J Pharmacol Exp Ther 2006; 319: 208-18; Drexler, et al., Anesthesiology 2006; 105: 297-304) or NMDA (Ogata, et al., J Pharmacol Exp Ther (2006) 318: 434-43; Dickinson, et al., Anesthesiology 2007; 107: 756-67) receptor can decrease responses to isoflurane, halothane, and other volatile anesthetics. Although mutations that render receptors insensitive to anesthetics could suggest a single site that is responsible for binding a specific drug, it need not be the case. Most of these mutations are believed to reside near lipid-water interfaces, either in amphiphilic protein pockets (Bertaccini et al., Anesth Analg (2007) 104: 318-24; Franks, et al., Nat Rev Neurosci (2008) 9: 370-86) or near the outer lipid membrane. It is possible that an anesthetic could be excluded from its protein interaction site because of size. However, it is also possible that the mutation substantially increases (but does not entirely exclude) the number of “non-specific” low-affinity anesthetic-protein interactions necessary to modulate the receptor. In this case, modulation of the mutant receptor will either only occur at anesthetic concentrations in excess of the wild-type minimum alveolar concentration (MAC) (Eger, et al., Anesthesiology (1965) 26: 756-63) or, if the drug is insufficiently soluble at the active site to allow a sufficient number of “non-specific” interactions with the mutant protein, no receptor modulation will be possible even at saturating aqueous drug concentrations.
Another argument for specific “induced fit” binding sites on ion channels is the “cut-off” effect. For example, increasing the carbon chain length of an alkanol increases lipid solubility and anesthetic potency, as predicted by the Meyer-Overton hypothesis (Overton C E: Studies of Narcosis. London, Chapman and Hall, 1991), until a 12-carbon chain length (dodecanol) is reached (Alifimoff, et al., Br J Pharmacol (1989) 96: 9-16). Alkanols with a longer chain length were not anesthetics (hence, a “cut-off” effect at C=13 carbons). However, the hydrocarbon chain length needed to reach the cut-off effect is C=9 for alkanes (Liu, et al., Anesth Analg (1993) 77: 12-8), C=2 for perfluorinated alkanes (Liu, et al., Anesth Analg (1994) 79: 238-44), and C=3 for perfluorinated methyl ethyl ethers (Koblin, et al., Anesth Analg (1999) 88: 1161-7). If size is essential to access a specific anesthetic binding site, then why is the “cut-off” chain length not constant? At the cellular level, straight-chain alcohols can maximally inhibit NMDA receptor function up to octanol with complete cut-off at C=10. But straight-chain 1, Ω-diols maximally inhibit NMDA receptors up to decanol, with complete cut-off not observed until C=16 (Peoples, et al., Mol Pharmacol (2002) 61: 169-76). Increasing hydrocarbon chain length does not only increase molecular volume, but also decreases water solubility. The cut-off effect therefore refers to a minimum water solubility necessary to produce an effect, rather than a maximum molecular size.
At the tens of micromolar concentrations or less, anesthetics most likely exert their effects on ion channels by specific binding to relatively high-affinity sites on proteins to induce a conformational change that alters ion conductance, either alone or in the presence of another endogenous ligand. However, these agents can still interact with other receptors (or the same receptor at different sites) if present in higher concentrations. For example, assume that two dissimilar receptors (R1 and R2) each can exert an anesthetic effect. Assuming that efficacy of a drug at R1=1, that R1 is able to produce a full anesthetic effect in isolation, and that the EC99 of R1 is less than the EC1 of R2, then this drug will produce anesthesia by selectively modulating R1. However, if any of these assumptions is not true, then some contribution of R2 will be required to produce an anesthetic effect (FIG. 1).
Many injectable anesthetics seem to follow the example described above. Propofol is a positive modulator of GABAA receptor currents with an EC50 around 60 μM (Hill-Venning, et al., Br J Pharmacol (1997) 120: 749-56; Prince, et al., Biochem Pharmacol (1992) 44: 1297-302; Orser, et al., J Neurosci (1994) 14: 7747-60; Reynolds, et al., Eur J Pharmacol (1996) 314: 151-6), and propofol is believed to mediate the majority of its anesthetic effects through potentiation of GABAA currents (Sonner, et al, Anesth Analg (2003) 96: 706-12). However, propofol also inhibits currents from the unrelated NMDA receptor with an IC50 of 160 μM (Orser, et al., Br J Pharmacol (1995) 116: 1761-8). Ketamine produces anesthesia largely through antagonism of NMDA receptors, which it inhibits with an IC50 of 14 μM (Liu, et al., Anesth Analg (2001) 92: 1173-81), although 365 μM ketamine also increases unrelated 4 transmembrane GABAA receptor currents by 56% (Lin, et al., J Pharmacol Exp Ther (1992) 263: 569-78). In these cases, it seems plausible that 2 different types of interactions (for high- vs. low-affinity responses) could occur on a single receptor to produce the same qualitative effect. In contrast, volatile inhaled anesthetics generally have little or no effect on GABAA and NMDA receptors at aqueous phase concentrations <50 μM (Lin, et al., J Pharmacol Exp Ther (1992) 263: 569-78; Moody, et al., Brain Res (1993) 615: 101-6; Harris, et al., J Pharmacol Exp Ther (1993) 265: 1392-8; Jones, et al., J Physiol (1992) 449: 279-93; Hall, et al., Br J Pharmacol (1994) 112: 906-10). It is possible that these agents are not specific ligands for any anesthetic-sensitive receptor that is relevant to immobility; thus they may rely only on nonspecific protein-ligand interactions that, in turn, may be reflected in the higher aqueous phase concentrations of these agents required for anesthesia (Table 1).